Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.
There are several important advantages of the horizontal slice preparation of the retina.
First, the morphology of the cells that expand dendrites horizontally, such as amacrine cells and horizontal cells, is preserved. It has been shown that there are over 20 types of amacrine cells in the retina (MacNeil and Masland, 1998), so that it is quite important to compare the morphology of the cells with the physiological property.
Second, in the whole-mount retina, the extensive coverage of glial cell s endfeets prevents the access of patch pipettes to amacrine cells. In the horizontal slices, the soma of amacrine cells is exposed to the surface of the slices.
Third, chemical reagents can easily reach to targeted cells and their dendrites, because these are located on the surface of the slices. Wash-in and wash-out of chemicals are fast and easy.
Fourth, fluorescent ion imaging such as conventional calcium imaging is performable. In the traditional vertical slices or the whole-mount retina, excitation light for imaging itself activates photoreceptors. In the horizontal slice preparation, photoreceptors and photopigments are totally removed so that there is no contamination of excitation light-evoked artifacts. In addition, the signal-to-noise ratio of imaging is much higher because the background activity of photoreceptors is eliminated. The biggest disadvantage of the horizontal slice preparation is that vertical connection between photoreceptors and ganglion cells is cut so that there is no visual information processing within the slice.
This technique is applicable to the retina of any standard animals such as goldfish, mouse, and rabbit (Fig.1B). The method of the horizontal slice may offer an alternative way in investigating the function of neurons and neural circuits in the retina.
The authors have nothing to disclose.
We thank Drs. Richard H. Masland and Akimichi Kaneko for giving us valuable advices and suggestions to establish the procedure of the horizontal slice preparation of the retina.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Agar | Sigma | A-1296 | ||
Agarose L | Wako | 317-01182 | Low-temperature melting agarose | |
Hyaluronidase | Sigma | Type I-S, 300 units/mg | ||
Medium | in mM: 102 NaCl, 3.1 KCl, 2.0 CaCl2, 1.0 MgCl2, 23 NaHCO3, and 10 glucose, maintained at pH 7.8, or, Ames’ Solution (Sigma) | |||
Microwave | ||||
Hot water bath | 35 C | |||
Vibratome slicer | Leica | VT1000S | ||
MF-Membrane filters | Millipore | HAWP01300 | Filter paper , pore size 0.45 um | |
Dissection tools | forceps, blade, pinch, twizzer, etc. | |||
Instant glue | eg., Alonalpha, Krazy Glu | |||
50 ml syringe | ||||
syringe filter | Millipore | SX0001300 |